CN116640743A - Endonuclease and application thereof - Google Patents
Endonuclease and application thereof Download PDFInfo
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- CN116640743A CN116640743A CN202310905132.8A CN202310905132A CN116640743A CN 116640743 A CN116640743 A CN 116640743A CN 202310905132 A CN202310905132 A CN 202310905132A CN 116640743 A CN116640743 A CN 116640743A
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
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Abstract
The invention provides an endonuclease mutant, comprising: (a) Compared with the amino acid sequence shown in SEQ ID NO.1, the endonuclease mutant contains at least one of the following sites: t43, G67, Q69, W89, Q99, G101, G132, S135, N136, F137, S140, N143, S145, F149, Q152, A154, Q169, N188 or G222, and a protein having an endonuclease activity function through amino acid mutation; or (b) the endonuclease mutant and the amino acid sequence shown in (a) are substituted and/or deleted and/or added by one or more conservative amino acid residues, and the amino acid sequence has the same function. The endonuclease mutant obtained by the invention has the capability of tolerating higher salt concentration and has higher activity in low-temperature and room-temperature environments.
Description
Technical Field
The invention relates to the technical field of nucleases, in particular to an endonuclease and application thereof.
Background
In recent years, with the development of biotechnology, more and more biological products such as antibodies, recombinant proteins, recombinant protein vaccines, viral vector vaccines, cell therapy and gene therapy drugs enter the therapeutic field, and the quality control of the biological products is also becoming stricter. Host nucleic acid residues present various biosafety risks, and excessive host nucleic acid residues may cause body infection, tumorigenesis, initiation of immune side reactions, gene transposition, recombination, etc., so that the host nucleic acid residues become a serious issue for quality control of biological products. The DNA residue in biological products expressed by yeasts and escherichia coli is definitely regulated in pharmacopoeia of China to be not more than 10 ng/dose. The pharmacopoeia of 2020 updates the DNA residual standard of rabies vaccine (Vero cells) for human to less than or equal to 3 ng per dose. In addition to the provision of residual amounts, the relevant guidelines issued by the FDA, CDE suggest that the residual cell host DNA fragment should not exceed the length of one functional gene (about 200 bp). Therefore, the step of removing nucleic acid must be included in the biological product production process and verified to ensure that the nucleic acid residues in the biological product meet the requirements of the corresponding regulations. The conventional methods for removing nucleic acid, such as precipitation, have problems in that aggregation and encapsulation occur, the removal efficiency is affected, and it is difficult to achieve the standard when the requirement for nucleic acid residues is high. In order to realize the control of the residual amount of nucleic acid in biological products, various nucleases have been widely used in the purification process of biological products in recent years and have good effects.
Nuclease is an enzyme capable of cleaving the phosphodiester bond of a polynucleotide chain, and belongs to a hydrolase that acts on the P-O position of the phosphodiester bond. The nucleases of different sources have different specificities and modes of action. Some nucleases can only act on RNA, called ribonucleases (rnases), some nucleases can only act on DNA, called deoxyribonucleases (dnases), and some nucleases have low specificity, and can act on both RNA and DNA, and therefore are called nonspecific nucleases. The nucleases can be further classified into exonucleases (exoucspases) and endonucleases (endonucleases) according to the positions of the nuclease action.
The currently prevailing commercial nucleases in the market are mainly Benzonase (serratia marcescens Serratia marcescens nuclease), omnicleave (Epicentre) or dnase I. Benzonase and Omnicleave are omnipotent nucleases that digest DNA and RNA, whereas DNase I digests only DNA. In general, nucleases have high nuclease activity at less than 100 mM NaCl, and when the salt concentration is higher than 100 mM, the enzyme activity is significantly reduced. Nucleases have a wide range of uses, for example, the main applications of Benzonase omnipotent nucleases are as follows: 1) Viral vaccines, viral vectors for cell and gene therapy, and removal of nucleic acids from oncolytic viruses; 2) Removing nucleic acid from biological products such as proteins; 3) Reducing the viscosity caused by the nucleic acid; 4) Cell culture medium can be added to prevent cell aggregation; 5) Reducing the viscosity of the E.coli cell lysate to improve filterability; 6) Pretreatment of particles (viruses, inclusion bodies, etc.); 7) ELISA, column chromatography, dielectrophoresis and western blot analysis, and the resolution and recovery rate can be improved after nuclease treatment. The nuclease can be used for efficiently removing the nucleic acid residues in biological products, but the activity of the currently mainstream nuclease is highly inhibited or even inactivated at higher salt concentration, and the requirement cannot be met. Therefore, the development of a salt-tolerant efficient nuclease has important significance for removing host nucleic acid residues in biological samples, and the nuclease with high salt tolerance can be widely applied to genetic engineering, biological macromolecule production, and particularly nucleic acid drug production. The commercial nuclease is generally expressed by yeast or supernatant of escherichia coli, the production process is complex, and the cost is high.
Tolerance of proteins to salt concentrations is a multifaceted factor. Wherein, the change of physicochemical properties of the protein surface can obviously influence the tolerance of the protein to the salt concentration, and the change trend of the protein surface comprises the reduction of isoelectric point, the reduction of hydrophobicity, the increase of heat capacity, the reduction of compressibility, the increase of flexibility parameters and the like.
Currently, sequence analysis of halophilic proteins has found a statistical increase in negatively charged amino acids in halophilic proteins. Enrichment of Asp and Glu results in a decrease in isoelectric point, while lysine is relatively decreased. Accumulation of acidic amino acids at the surface is the most prominent feature of enzymes that accommodate high salinity, and this increase in acidity is also believed to prevent protein aggregation itself. In addition, the addition of acidic amino acids also promotes protein solubility, and repulsive forces between negative charges provide flexibility for protein function at high salt concentrations. The research shows that after the acid amino acid substitution is carried out on the surface of carbonic anhydrase II and choline kinase, the salt tolerance is obviously improved.
However, in addition to the increase in acidic amino acids on the protein surface, an increase in basic amino acids has also been reported to enhance the salt tolerance of the protein. In seawater with a salinity of 3.5%, about 470 mM Na was contained + And 540 mM Cl - . Thus, most marine prokaryotes can be considered to be slightly halophilic organisms, and their periplasmic/extracellular proteins are exposed to the outside higher salt concentration environment. Vibrio belongs to gram-negative bacteria, is mainly distributed in river and lake outlets and marine environments, grows well in environments with salinity of 2-4%, and cannot grow in salt-free environments. It was found that endonuclease I (VsEndA) derived from the marine bacterium vibrio salmonicus exhibits an adaptability to higher salt concentrations than homologous endonuclease I (VcEndA) derived from the salt water bacterium vibrio cholerae. VsEndA has the highest nuclease activity at a salt concentration of 425 mM NaCl, and still has about 60% activity at a salt concentration of 600 mM. In contrast, vcEndA only had the highest activity at 200 mM salts, with only 10% of VcEndA activity at 500 mM salts. The protein structure of VsEndA is highly similar to that of VcEndA, but the positively charged amino acids on the surface of VsEndA are significantly increased, for exampleSuch as lysine and arginine. Researchers speculate that an increase in the positive surface charge of VsEndA can be correlated with Cl in high salt environments - Interactions to stabilize the structure of the protein itself, and furthermore the more positive charges on the VsEndA surface are also favourable for interactions with negatively charged DNA.
Although VsEndA exhibits good salt tolerance, the enzyme activity drops dramatically after salt concentration in solution exceeds 500 mM, with only about 30% of the enzyme activity at 700 mM salt concentration, and still the high salt tolerance requirements of the biologic production are not met. Thus, there is a need for a nuclease with better salt tolerance.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides endonuclease and application thereof. The method solves the problems that the salt tolerance is not high and the high salt tolerance requirement of biological product production cannot be met in the prior art.
In one aspect of the invention, there is provided an endonuclease mutant comprising: (a) Compared with the amino acid sequence shown in SEQ ID NO.1, the endonuclease mutant contains at least one of the following sites: a protein having an endonuclease activity function, wherein T43, G67, Q69, W89, Q99, G101, G132, S135, N136, F137, S140, N143, S145, F149, Q152, a154, Q169, N188, or G222 is subjected to an amino acid mutation; or (b)
(b) The endonuclease mutant and the amino acid sequence shown in the (a) are subjected to substitution and/or deletion and/or addition of one or more conservative amino acid residues to obtain the amino acid sequence with the same function.
Further, the endonuclease mutant comprises at least one site substitution selected from the group consisting of: T43E, G67E or G67K, Q69 8239D, Q99K, G101K, S K, N136R, F137E, S140E, N D or N143K, S145D, F39149 152K, Q169D, N E, G E or G222K.
Further, the endonuclease mutants include any one of the amino acid mutations shown below:
G67E+Q69E+S140E+N143D+S145D+F149D+Q169D+N188E+G222E；
Q69E+S140E+N143D+G222E；
G67K+N143K+Q152K+G222K；
T43E；
F137E；
W89D+N136R；
Q99K+G101K+S135K。
in a second aspect of the invention, there is provided a DNA molecule encoding the endonuclease mutant described above.
Further, the nucleotide sequence of the DNA molecule corresponds to the amino acid sequence shown in SEQ ID NO. 2-7.
In a third aspect of the invention, there is provided a recombinant vector, expression cassette or host cell comprising a DNA molecule as described above.
In a fourth aspect of the invention, there is provided a kit comprising the endonuclease mutant described above.
In a fifth aspect of the invention, there is provided a method of cleaving nucleic acid using the endonuclease mutant, DNA molecule, recombinant vector, expression cassette or host cell described above.
In a sixth aspect of the present invention, there is provided a method of producing the above endonuclease mutant, culturing the above-described host cell, and obtaining the endonuclease mutant from the culture; preferably, the host cell comprises E.coli.
In a seventh aspect, the present invention provides the use of the endonuclease mutant, the DNA molecule, the recombinant vector, the expression cassette or the host cell described above, or the kit described above for removing residual nucleic acid from a biological product.
The term "biological product" refers to a biological material prepared using microorganisms, cells and tissues and fluids of various animal and human origin, and the like, which are common or obtained by biotechnology such as genetic engineering, cellular engineering, protein engineering, fermentation engineering, and the like, and includes bacterins, vaccines, toxins, toxoids, immune serum, blood products, immunoglobulins, antigens, allergens, cytokines, hormones, enzymes, fermentation products, monoclonal antibodies, DNA recombinant products, in vitro immunodiagnostic products, and the like.
The term "kit" refers to a cartridge composed of chemical reagents that can be used to detect chemical components, drug residues, virus species, and the like.
Since the first 21 amino acids of the wild-type VsEndA protein are signal peptides, the protein synthesized in the organism will automatically cleave the signal peptide, so that upon recombinant expression, the expression is started from position 22, in the present invention SEQ ID No.1 is the VsEndA protein cleaved from the signal peptide, and thus the first amino acid is numbered from position 22.
The technical principle of the invention is as follows: the invention is based on a computer aided design method, and is modified under the condition of not affecting the overall structural stability of the VsEndA, and a plurality of polar amino acid substitutions are carried out at specific sites on the surface of the VsEndA, so that the hydrophobicity and the electroneutrality of the surface of the VsEndA are reduced. Through virtual screening and experimental final verification of a computer, reasonable amino acid substitution changes the surface electrification, hydrophobicity and stability of the protein, so that the VsEndA has the capability of tolerating higher salt concentration and has higher activity in low-temperature and room-temperature environments. In addition, the nucleases of the present invention are expressed in E.coli in inclusion body form and the final product is obtained by directed mutagenesis technique.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention obtains novel nuclease with high salt tolerance and low temperature activity by carrying out point mutation on VsEndA. The nuclease obtained by screening reaches the highest enzyme activity in the NaCl environment of 700 mM, can keep higher nuclease activity in the NaCl environment of 400-900 mM, and basically covers the high-salt environment in the production process of biological products. The modified molecule still maintains higher biological activity in low temperature and room temperature environment, reaches the maximum value of enzyme activity at 25 ℃, and is also just suitable for the environmental temperature of biological product production.
(2) The novel nuclease is expressed as inclusion bodies by escherichia coli, and is purified by a protein renaturation method to obtain the high-activity nuclease, so that the production process is simple, the production cost is low, and the cost advantage is achieved.
(3) The nuclease is a novel omnipotent nuclease, not only acts on DNA, but also acts on RNA, and the substrate has universality and has huge economic benefit.
Drawings
FIG. 1 is an alignment chart of amino acid sequences and three-dimensional structures of three homologous nucleases in example 1 of the present invention;
FIG. 2 is a surface potential analysis chart of VsEndA in example 1 of the present invention;
FIG. 3 is an E.coli expression electrophoresis chart of VsEndA and its mutant in example 2 of the present invention, wherein T represents whole bacteria, S represents supernatant, and P represents pellet;
FIG. 4 is an electrophoresis chart of VsEndA and its mutant pure product in example 2 of the present invention;
FIG. 5 is a graph showing the results of enzyme activity detection of different nucleases at different salt ion concentrations in example 3 of the present invention;
FIG. 6 is a graph showing the results of enzyme activity detection of 1527 in example 4 of the present invention at different temperatures;
FIG. 7 is a graph showing the cleavage effect of 1527 on DNA and RNA in example 5 of the present invention.
Detailed Description
The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.
Example 1 computer aided design based protein structural engineering and mutant screening
1. Screening of halophilic microorganisms
(1) Alignment of amino acid sequences and three-dimensional structures of different halophilic microorganisms
Three different vibrio halophilus nucleases VnnEndA (vibrio vulnificus), vcEndA (Vibrio cholerae) and VsEndA (Vibrio salmonicida) were selected as subjects for amino acid sequence alignment and protein three-dimensional structure alignment of three endonucleases i, and the results are shown in fig. 1.
As can be seen from FIG. 1, the homologous sequence alignment was performed with Clustal Omega on three nucleases, which have a high degree of sequence homology (homology up to 75%) with key amino acids associated with metal ion interactions and activities remaining identical. The three-dimensional structures have high similarity, the whole structure is in a sphere shape with a notch, and the notch area is the enzyme activity center. The three nuclease structures are all relatively rigid and contain a large number of alpha helices and two pairs of small beta sheets, four pairs of disulfide bonds. The enzyme activity center is located in a long and narrow region composed of alpha helices and beta sheets, and the metal ions are also located therein. Taking VsEndA as an example, conserved His84 and Arg103 are critical to enzyme activity. In particular, five pairs of salt bonds, asp40-Arg216, glu81-Arg76, glu83-Arg134, glu117-Arg103 and Glu210-Arg176, are highly conserved among three types of vibrio salina nucleases, and have important significance in maintaining the stability of the overall structure of the nuclease.
(2) Surface amino acid alignment of three nucleases
Analysis of the surface amino acids of the three nucleases in PyMOL revealed that the surface of VsEndA had a large number of basic amino acid substitutions compared with the other two nucleases, so that the surface of VsEndA had a large number of positive charges, which on the one hand helped to promote the solubility of VsEndA and inhibit self aggregation, and on the other hand helped to bind VsEndA to DNA and Cl in high salt solution - The interaction maintains its own structure stable.
Because the salt tolerance of VsEndA is significantly better than that of the other two nucleases, vsEndA was selected as a study subject and further modified.
2. Modification of VsEndA
VsEndA was subjected to point mutation by reducing the hydrophobicity and electroneutrality of the protein surface.
(1) Surface potential analysis of VsEndA
The surface potential analysis of VsEndA is shown in fig. 2. As can be seen from FIG. 2, the active center region of VsEndA is rich in lysine and arginine, and has a strong positive charge, which facilitates the approach of negatively charged DNA to the active center. The back region of the active center presents a number of negatively charged acidic amino acids,is beneficial to Na in high-salt environment + Interaction promotes stability of the overall structure. The distribution of the VsEndA surface charge has obvious biplanarity, namely, the active center is positively charged, the inactive center is negatively charged, and the wide reconstruction space is provided.
(2) Selection of amino acid mutation sites of VsEndA
In order not to influence the enzyme activity and structural stability of the VsEndA, a flexible region far away from an active center is selected for transformation when the VsEndA is transformed, and the sites such as T43, G67, Q69, W89, Q99, G101, G132, S135, N136, F137, S140, N143, S145, F149, Q152, A154, Q169, N188 and G222 are finally selected for mutant screening by combining the region with relatively low conservation in homologous sequence alignment.
(3) Screening of VsEndA mutants
When selecting mutants, reasonable polar amino acid substitution is carried out on different mutation sites, and a plurality of different mutants are designed. Firstly, simulating the change of free energy after mutation by using a computer, and selecting mutants with smaller or slightly raised free energy after mutation so as to reduce the influence of amino acid substitution on the overall stability of the structure. And further adopting a homologous modeling method to simulate three-dimensional structures of different mutants respectively, and optimizing the predicted structural conformation in a certain force field. And finally, carrying out molecular docking on each mutant and DNA from the VnnEndA crystal structure (PDB ID:1 OUP) by adopting a molecular docking method, and selecting the mutant which is reasonable in spatial conformation and high in affinity and is combined with the DNA. The selected VsEndA mutants are shown in table 1.
TABLE 1 amino acid mutation information for different mutants
Example 2 construction of VsEndA and mutant plasmids therefor, protein expression and purification
1. Plasmid construction
In this patent, 7 different mutants were obtained after combining the various mutations as in table 1 for wild-type VsEndA, 1525 (Q69E, S140E, N143D, G222E), 1526 (G67E, Q69E, S140E, N143D, S145D, F149D, Q169D, N188E, G222E), 1527 (G67K, N143K, Q152K, G222K), 1692 (T43E), 1699 (F137E), 1700 (W89D, N136R) and 1701 (Q99K, G101K, S135K), respectively, and specific amino acid sequence information for each molecule is shown in table 2. The first 21 amino acids from the marine bacterium vibrio salmon VsEndA are signal peptides, so the wild type VsEndA synthesized in this example starts with alanine 22.
The gene sequences of VsEndA (hereinafter labeled WT) and mutants thereof (1525, 1526, 1527, 1692, 1699, 1700, 1701) were delegated to beijing, department of biotechnology, ltd. The target fragment was obtained by PCR according to the procedure described in molecular cloning. And then the target fragment is recombined and connected with a general vector pET28a, transformed, sequenced and preserved, so that a plasmid capable of expressing the corresponding protein is obtained. Plasmid preparation was performed on the endonuclease according to the procedure described in Qiagen Mini-prep Kit.
TABLE 2 Endonuclease amino acid sequence listing
2. Protein expression
The plasmid constructed successfully was transformed into E.coli expression strain BL21 (DE 3), the monoclonal colony was picked from the plate and transferred into a triangular flask containing Kana-resistant 500 mL TB medium at a volume ratio of 1:50, the initial OD600 was about 0.1, 37℃at 220 rpm, the culture was carried out until the OD600 was 2.0, IPTG was added at a final concentration of 0.5 mM, and after culturing at 37℃at 220 rpm, 4 h, the strain was harvested. Protein expression was detected using SDS-PAGE, as shown in FIG. 3.
As can be seen from FIG. 3, all proteins were successfully expressed in the form of inclusion bodies (inclusion bodies), and the expression amount was high.
3. Thallus crushing recovery inclusion body
The thalli are crushed under high pressure, and the inclusion bodies are recovered after washing, so that preparation is made for the next protein renaturation. The buffer used for cell disruption was 20 mM Tris, pH 8.0, 0.5M NaCl.
4. Protein purification
(1) Protein renaturation
The inclusion bodies in step 3 were solubilized at room temperature using 10 times (v/w) volume of denaturing solution (8M Urea, 50 mM Tris, pH 9.0), and after 30 minutes DTT was added and the reduction continued for 30 minutes. The denatured solution was centrifuged using 12000-g, and the supernatant was added dropwise to 50 volumes of renaturation solution (20 mM Tris, 0.5M arginine, 2.5 mM Cysteine, pH 8.0), stirred overnight at room temperature, and the renaturation effect was detected by the second-day mass spectrometry.
(2) Protein purification
The pH of the renatured protein in the step (1) is adjusted to 7.5 by hydrochloric acid, the conductivity is diluted to below 10 ms/cm by deionized water, and the supernatant is directly captured by passing through an SPHP column (GE company).
The operation is as follows: the SPHP column was equilibrated with 5 column volumes of equilibration solution (20 mM Tris, pH 7.5) prior to purification; passing the renaturation solution through a column, and then washing the column by using a balancing solution with the volume of 5 times of the column to remove non-specific binding proteins; the enzyme solution was obtained by eluting with a linear gradient of 15 column volumes of elution buffer (20 mM Tris, pH 7.5, 1M NaCl) and collecting the eluate containing the target protein.
As shown in FIG. 4, after SPHP purification, the 8 target proteins were clearly banded, with higher purity, only with a slightly poorer yield and purity of 1526.
Example 3 mutant enzyme Activity detection at different salt concentrations
The nuclease activity of each mutant was tested at different salt ion concentrations using commercially available Benzonase and WT as controls. The specific detection method comprises the following steps:
first, calf thymus DNA was dissolved in 5 mM MgCl 2 The final concentration was 0.1 mg/ml in 0.1 mg/ml BSA, 50 mM Tris, pH 8.0. A DNA sample of 2.5. 2.5 ml was taken, a certain amount of NaCl and 0.125. 0.125 ml of the enzyme solution obtained in example 2 were added according to the set salt concentration, and 0.125 ml of 1 XPBS was added to the control group, and after mixing, it was placed in a 37℃water bath and sampled at 15 min, 30 min, 45 min and 60 min, respectively. Taking after each step of reaction0.5 ml of the reacted product was added with 0.5. 0.5 ml% perchloric acid solution, and after mixing, incubated on ice for 30 min.14000 After centrifugation of the final reaction at rpm, the OD of the supernatant was measured at 260 nm (uv spectrophotometer) and the enzyme activity was calculated.
The calculation formula is as follows:
DeltaA. Absorbance of the solution at time t was measured. t: measuring reaction time of solution
As shown in FIG. 5, benzonase has high nuclease activity under the condition of less than 100 mM NaCl, the enzyme activity is obviously reduced when the salt concentration is higher than 100 mM, and the Benzonase is basically inactive when the salt concentration is higher than 300 mM. In contrast, WT showed the strongest nuclease activity at a salt concentration of 400 mM, with the enzyme activity gradually decreasing as the salt concentration in the reaction system increased. 1525 and 1527 exhibit better salt tolerance, 1525 has higher nuclease activity in the salt concentration range of 400-800 mM, and the optimum salt concentration is 600 mM, while 1527 has higher activity in the salt concentration range of 500-800 mM, and the optimum salt concentration is 700 mM. After transformation, the tolerance of 1525 and 1527 to NaCl is obviously improved, and the salt tolerance requirement of biological products in production is met.
Example 4 Activity of enzymes at different temperatures
In actual biological production processes, the biological is typically in a room temperature environment. The nuclease activity at room temperature significantly affects the nucleic acid residues of the final product. Conventional nucleases generally have higher enzymatic activity at 37-42 ℃ and greatly reduce enzymatic activity in room temperature environments. Based on the actual production requirements, 1527 nuclease activity at a lower temperature was detected in this example, the experimental NaCl concentration was chosen to be 700 mM, and the experimental method was substantially the same as in example 3. The experimental results are shown in FIG. 6.
From the results, 1527 was stable at low temperature, and a certain nuclease activity was maintained. With increasing temperature, 1527 exhibited the highest enzyme activity at 25 ℃ (near room temperature), further increasing the reaction temperature and gradually decreasing the activity of 1527. Therefore, 1527 has the highest activity under the room temperature condition, meets the actual production requirement, and is beneficial to large-scale application in industrial production.
Example 5 1527 nuclease Activity on DNA and RNA
At 5 mM MgCl 2 In a reaction system of 0.7M NaCl, 20 mM Tris, pH 8.0, 5. Mu.g DNA and 5. Mu.g RNA were digested with 1527. After 30 minutes of reaction at room temperature, the cleavage results were detected by electrophoresis on a 1% agarose gel. The results are shown in FIG. 7.
From the results, it was found that the DNA and RNA were completely enzymatically cleaved off at 1527 as compared with the control group. 1527 nuclease is a totipotent enzyme and has wider application range.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (11)
1. An endonuclease mutant comprising: (a) Compared with the amino acid sequence shown in SEQ ID NO.1, the endonuclease mutant contains at least one of the following sites: t43, G67, Q69, W89, Q99, G101, G132, S135, N136, F137, S140, N143, S145, F149, Q152, A154, Q169, N188 or G222, and a protein having an endonuclease activity function through amino acid mutation; or (b)
(b) The endonuclease mutant and the amino acid sequence shown in the (a) are subjected to substitution and/or deletion and/or addition of one or more conservative amino acid residues to obtain the amino acid sequence with the same function.
2. An endonuclease mutant according to claim 1 wherein: the endonuclease mutant comprises at least one substitution at a position selected from the group consisting of: T43E, G67E or G67K, Q69E, W D, Q99K, G K, S K, N3749K, N R, F E, S140E, N D or N143K, S145D, F149D, Q152K, Q D, 188E, G222E or G222K.
3. An endonuclease mutant according to claim 2 wherein: the endonuclease mutants include any of the amino acid mutations shown below:
G67E+Q69E+S140E+N143D+S145D+F149D+Q169D+N188E+G222E；
Q69E+S140E+N143D+G222E；
G67K+N143K+Q152K+G222K；
T43E；
F138E；
W89D+N136R；
Q99K+G101K+S135K。
4. a DNA molecule encoding the endonuclease mutant of any one of claims 1 to 3.
5. The DNA molecule of claim 4, wherein the nucleotide sequence of said DNA molecule comprises the nucleotide sequence set forth in SEQ ID nos. 2-7.
6. A recombinant vector, expression cassette or host cell comprising the DNA molecule of claim 4.
7. A kit comprising the endonuclease mutant of any one of claims 1-3.
8. A method for cleaving nucleic acid, characterized in that the endonuclease mutant according to any one of claims 1 to 3, the DNA molecule according to claim 4, the recombinant vector, the expression cassette or the host cell according to claim 5 is used.
9. A method for preparing the endonuclease mutant according to any one of claims 1 to 3, characterized in that the host cell according to claim 6 is cultivated and the endonuclease mutant is obtained from the culture.
10. The method of claim 9, wherein the host cell comprises e.
11. Use of the endonuclease mutant of any one of claims 1 to 3, the DNA molecule of claim 4 or 5, the recombinant vector, expression cassette or host cell of claim 6, the kit of claim 7 for removing residual nucleic acid from a biological product.
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